Electrotherapy device using low frequency magnetic pulses

ABSTRACT

An apparatus and method for treating a disorder selected from the group of physiological, neurological and behavioral disorders, the method comprising applying to a subject a specific low frequency pulsed magnetic field (Cnp) having a plurality of intermittent waveforms, for a time effective to produce a desired effect in a target tissue.

This application of PCT/CA97/00388 filed Jun. 5, 1997 and also claimsthe benefit of provisional no. 60/019,184 filed Jun. 6, 1996.

FIELD OF THE INVENTION

This invention relates to magnetic fields and in particular, to the useof specifically designed low frequency pulsed magnetic fields (Cnps) formodifying a variety of clinical physiological and neurological behaviorsand conditions in vertebrates and invertebrates.

BACKGROUND OF THE INVENTION

Diverse studies have shown that the behavioral, cellular andphysiological functions of animals can be affected by magnetic stimuli.Weak magnetic fields exert a variety of biological effects ranging fromalterations in cellular ion flux to modifications of animal orientationand leaming, and therapeutic actions in humans. A number of magneticfield exposures have been shown to reduce exogenous opiate (e.g.morphine) and endogenous opioid peptide (e.g. endorphin) mediatedanalgesia in various species, including humans (Kavaliers & Ossenkopp1991; Prato et al., 1987; Betancur et al., 1994; Kavaliers et al., 1994;Del Seppia et al., 1995; and Papi et al., 1995). As well, extremely lowfrequency (ELF) magnetic field exposures are reported to modify homingpigeon behavior (Papi et al., 1992) and spatial learning in rodents(Kavaliers et al., 1993, 1996) in a manner consistent with alterationsin opioid function.

There are several theories addressing the mechanism of the effect of lowfrequency magnetic field exposure on tissues. For example, low frequencymagnetic field exposures have been proposed to exert their effect(s)through the induction of electric currents (Polk 1992; and Weaver &Astumian 1990). Weak magnetic fields have also been proposed to bedetected by particles of magnetite in tissue and by virtue of thisdetection have a physiological effect (Kirschvink & Walker 1985);however, this magnetite based mechanism is not widely believed (Prato etal., 1996).

Extremely low frequency (ELF) magnetic fields are a physical agent whichhave little attenuation in tissue and therefore, can be used to alterendogenous processes provided they can be detected and their detectioncan be coupled to a physiological process. It is now shown that magneticfields may be designed as time varying signals such that they can beused to alter specific targeted physiological processes and in thismanner can be used to treat/modify various neurological andphysiological conditions and behaviors. It was therefore an object ofthe present invention to provide novel specific low frequency pulsedmagnetic fields having a plurality of intermittent wavefonns for use totreat a variety of physiological, neurological and behavioral disordersin both vertebrates and in invertebrates.

SUMMARY OF THE INVENTION

The applicants have now designed and characterized complex low frequencypulsed magnetic fields (Cnps) and their effects on physiological,neurological and behavioral conditions. The low frequency pulsedmagnetic fields are specifically designed to target and alter complexneuroelectromagnetic applications and permit the development oftherapeutic strategies in order to treat and/or alter variousphysiological, neurological and behavioral disorders.

Broadly stated, the present invention relates to complex low frequencypulsed magnetic fields (Cnps) which are designed and used as atherapeutic treatment for disorders and behaviors including: alleviationof pain and anxiety; restoration of balance; improved leaming; treatmentof epilepsy; and depression; and for moderating eating habits.

In accordance with an aspect of the present invention there is provideda therapeutic method for treating physiological, neurological andbehavioral disorders, the treatment comprising: subjecting a mammal to aspecific low frequency pulsed magnetic field having a plurality ofwaveforms designed with a length and frequency relative to the targettissue intermittent with a built-in variable latency period and a fixedrefractory period, for a time effective to produce a desiredphysiological effect.

The method of the present invention if not completely, at leastpartially, averts the development of tolerance which is typical withrepeated administrations of analgesic drugs and in particular, opioids.The method also decreases the need to use pharmacological agents totreat and alleviate various physiological, neurological and behavioralconditions. In addition, the low frequency pulsed magnetic fields can bedesigned with specific waveforms to target specific tissues to affectdifferent physiological functions without presentation of unwanted sideeffects.

Other objects, features and advantages of tie present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIG. 1 shows a specific low frequency pulsed magnetic field (Cnp) usedto induce analgesia.

FIG. 2 shows detail of two of the waveforms of FIG. 1. Comparison of thex-axis between FIG. 1 and FIG. 2 shows the portion of the time axiswhich has been expanded. Sub-label 1 corresponds to the waveform andsub-label 2 to the latency period.

FIG. 3 shows a Cnp designed to target the vestibular system of rodents.The top panel corresponds to Cnp in time and the lower panel correspondsto the magnitude of the Fourier Transform of the Cnp.

FIG. 4 shows in detail three waveforms of the vestibular Cnp shown inFIG. 3. As in FIG. 2, the x-axis indicates that portion of the time axiswhich has been expanded and relates FIG. 3 to FIG. 4. Sublabel 1corresponds to the waveform and sub-label 2 to the latency period.

FIG. 5 shows detail of the Cnp used to target the human vestibularsystem. There are differences in the refractory period as compared tothe Cnp targeted for rodents (See FIG. 3). The top panel corresponds toCnp in time and the lower panel to the magnitude of the FourierTransform of the Cnp.

FIG. 6 shows the effect of a Cnp targeted to induce analgesia in a landsnail. The y-axis corresponds to a measure of analgesia. Basalcorresponds to measurements done prior to exposure. The induction ofanalgesia is only thirty percent when a simple magnetic field waveformis applied (30 Hz sinusoidal 15 minute continuous exposure with a peakamplitude of 190 μT and a static field of 76 μT parallel to the 30 Hzfield). However when a specific designed magnetic field pulse (Cnp Exp)is given the analgesia is increased by more than 100 percent from theBasal value.

FIG. 7 shows a Cnp pulse designed to target central nervous systemtargets which should increase analgesia in land snails. The upper panelcorresponds to Cnp in time and the lower panel to the magnitude of theFourier Transform of the Cnp.

FIG. 8 shows that oploid antagonists reduce, but do not block, Cnpinduced analgesia.

FIG. 9 shows the effect of a complex neuroelectromagnetic pulse (Cnp),designed to increase analgesia, consisting of a series of time-varyingextremely low frequency components (<300 Hz) (A) repeated betweenrefractory periods of several seconds (B). The Cnps are repeated for thelength of the exposure period (15 or 30 mins) (C). Peak Cnp exposure isset at 100 μT vertical, with the horizontal and vertical static magneticfield set to counter the Earth magnetic field. Sham exposures consistedof a three-dimensionally (3-D) zeroed Earth magnetic field (3 orthogonalnested Helmholtz coils tuned to oppose the Earth magnetic field towithin ±0.1 μT, horizontal component =14.7 μT, vertical component 43.3μT.)

FIG. 10 shows the effects of an acute 15 or 30 min exposure to either aspecific pulsed magnetic field (Cnp) or sham exposure condition on thethermal (40° C.) response latencies of individual hydrated snails(N=120). Response latencies were recorded prior to (Pre) and afterexposure. Sham 15 and 30 min exposures were not significantly differentand were combined. Error bars represent the Standard Error of the Mean(SEM), and where not visible are embedded within the symbol.

FIG. 11 shows the effects of either (A) 15 min or (B) 30 min dailyrepeated exposures to either a specific pulsed magnetic field (Cnp) orsham exposure condition on the thermal (40° C.) response latencies ofindividual hydrated snails (N=60).

Response latencies were recorded prior to (Pre) and after (0, 15, 30, 60min) exposure. Response latencies from days 1, 3, 6 and 9 are shown.There were no significant differences within the sham groups, hence thegroups were collapsed. Error bars represent the Standard Error of theMean (SEM), and where not visible are embedded within the symbol.

FIG. 12 shows the effects of either (A) 15 min or (B) 30 min dailyrepeated exposures to either a specific pulsed magnetic field (Cnp) or(C) sham exposure condition on the thermal (40° C.) response latenciesof individual hydrated snails (N=60), shown in 3-D perspective. Responselatencies were recorded prior to (Pre) and after (0, 15, 30, 60 min)exposure. There were no significant differences within the sham exposureor pre-exposure latencies.

FIG. 13 shows the effects of (A) 15 and (B) 30 min daily repeated acuteexposure to a sham or specific pulsed magnetic field (Cnp) on thethermal (40° C.) response latencies (I 5 min post-exposure) ofindividual hydrated snails (N=60). Day 10 records the effects ofcondition reversal; in that, the previously sham exposed groups wereexposed to the Cnp, and vice versa. Error bars represent the StandardError of the Mean (SEM), and where not visible are embedded within thesymbol.

FIG. 14 shows thermal (40 ° C.) response latencies of snails (N=60)exposed to a specific pulsed magnetic field (Cnp) or sham condition for15 or 30 min daily for 9 consecutive days. Response latencies weretested on days I and 9 prior to (Pre) and after (0, 15, 30, 60 min)exposures. There were no significant differences within the sham groups,hence the groups were collapsed. Error bars represent the Standard Errorof the Mean (SEM), and where not visible are embedded within the symbol.

FIG. 15 shows thermal (40° C.) response latencies of individual snails(N=30) exposed for 15 min to either a specific pulsed magnetic field(Cnp) or sham magnetic field for 9 consecutive days (normal). On day 10the snails were exposed to the Cnp or sham condition while under a novelenvironment condition (novel). Response latencies were tested prior to(Pre) and after (0, 15, 30, 60 min) exposure. Error bars represent theStandard Error of the Mean (SEM), and where not visible are embeddedwithin the symbol. (*P<0.01 , **P<0.001)

FIG. 16 shows thermal (40° C.) response latencies of individual snails(N60), that had been exposed for 15 min daily for 9 consecutive days toeither a sham or Cnp. Response latencies were tested on day 10 prior to(Pre) and after being injected either with the δ opiate agonist, DPDPE,(0.05 μg/1.0 μl saline) or saline vehicle (1.0 μl) at 15, 30, 60 minintervals. Error bars represent the Standard Error of the Mean (SEM),and where not visible are embedded within the symbol.

FIG. 17 shows Cnp induced activity in Deer mice. The Cnp shown in FIG. 3was used.

FIG. 18 shows Cnp generated interference of human standing balance. TheCnp shown in FIG. 5 was used.

FIG. 19 shows the number of rearing behaviors in deer mice in each 5 minsegment of the 10 min exposure. A rearing behavior is counted when theanimal rears up on the hind limbs without touching any of the outsidewalls of the exposure container. The Cnp (see FIG. 3) exposure producedsignificantly greater counts than either the sham or 60 Hz exposure. Thefirst (0-5) and second (6-10) minute Cnp segments are not significantlydifferent. There are no significant differences within or between thesham and 60 Hz exposures. Error bars represent the standard error of themean in Cnps.

FIG. 20 shows the overall effect of Cnp (see FIG. 3) on the rearingbehavior in deer mice.

FIG. 21 shows the number of centerline crossings in each 5 min segmentof the 10 min exposure. A centerline crossing is counted when the entireanimal traverses across the center of the exposure container. The Cnpexposure produced significantly greater counts than either the sham or60 Hz exposure. The first (0-5) and second (6-10) minute Cnp segmentsare significantly different. There are no significant differences withinor between the sham and 60 Hz exposures. Error bars represent thestandard error of the mean.

FIG. 22 shows the overall effect of the Cnp of FIG. 3 on the centerlinecrossing activity of deer mice.

FIG. 23 shows the number of climbing movements in each 5 min segment ofthe 10 min exposure. A climbing movement is counted when the animalattempts to climb or reach up the side of the exposure container with 2or more limbs extended off the floor and ends when all four limbs are onthe floor. The Cnp exposure of FIG. 3 produced significantly greatercounts than either the sham or 60 Hz exposure. The first (0-5) andsecond (6-10) minute Cnp segments are significantly different. There areno significant differences within or between the sham and 60 Hzexposures. Error bars represent the standard error of the mean.

FIG. 24 shows the overall effect of the Cnp of FIG. 3 on the number ofclimbing movements in deer mice.

FIG. 25 shows the overall effect of the Cnp of FIG. 3 on the totalduration of grooming behaviors in deer mice.

FIG. 26 shows the sucrose preference, expressed as the percent ofsucrose drunk out of the total fluid intake, in male and femalereproductive deer mice. Percents are referred to the day of pairing withLithium Chloride or saline solution, the 3 days following pairing andthe two re-test days (10 days after recovering from sucrose aversion).

FIG. 27 shows the total fluid intake of male and female deer mice beforeand after treatment with Lithium Chloride or Saline Solution.

FIG. 28 shows the total fluid intake of male and female deer mice afterpairing of the apple juice with a Cnp or a sham magnetic field.

FIG. 29 shows the target taste (apple juice or sucrose) preference,expressed as the percent of target fluid drunk out of the total fluidintake, by male and female deer mice after exposure to the Cnp or a shammagnetic field.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As hereinbefore mentioned, the present invention provides designed andcharacterized low frequency pulsed magnetic fields (Cnps) which havespecific effects on physiological, neurological and behavioralconditions in vertebrates and invertebrates. The specific low frequencymagnetic fields are designed for complex neuroelectromagneticapplications and permit the development of therapeutic strategies inorder to treat and/or alter various physiological, neurological andbehavioral disorders particularly in mammals and more specifically inhumans.

Magnetic fields have been demonstrated to have various biologicaleffects in humans, rodents and snails. Such magnetic fields can bedetected and this detection can be broadly linked to certainphysiological processes. It is now demonstrated that low frequencypulsed magnetic fields can be designed specifically to alter specifictargeted physiological processes and in this manner provide atherapeutic method for treatment and alleviation of certain conditionswithout the need for pharmacological intervention which is expensive andwhich poses several problems with respect to side effects of certaindrugs.

In the present invention it is now demonstrated that the magnetic fieldexposure must be designed as a time varying signal such that it can beused to alter a specific targeted physiological process. The designedlow frequency pulsed magnetic field (Cnp) is valid independent of thedetection mechanism. However, different detection mechanisms may affecthow the Cnp is scaled and how it is delivered. Under certain conditionsextremely low frequency (ELF) magnetic fields can be detected directlyaccording to a resonance model. If the tissue exposed with the Cnp pulsedetects magnetic fields by the resonance model, then the amplitude ofthe Cnp pulse and possible DC (direct current) offsets are important andmust be specified with limits below which and above which effects willlessen. On the other hand, if magnetic field detection is indirect, ie.the ELF magnetic fields are detected by tissue-induced electromotiveforce (i.e. Faraday's Law of Induction) then the effects will have alower threshold below which no effect will be seen and then above thisthreshold effects will increase. However, even for indirect detection, amaximum threshold will exist above which the induced currents (caused bythe induced EMF) will be so great that targeted effects will be swampedby large unwanted side effects. Therefore, different detectionmechanisms might affect amplitude and DC offset of the Cnp, but thegeneral design rules will not change. Individual features of ageneralized Cnp are shown in FIG. 1, table 1 and FIG. 2 and are labeledin letters running from a. How these features are specified and targetedto a certain physiological/behavioral effect is described below.

Table 1 represents the 8-bit digital analog values of the specificpoints used in the construction of FIG. 1 (Cnp used to induceanalgesia). The columns are contiguous, that is, they are essentiallyone long column representing all of the serial points of FIG. 1. Thevalues presented in this table can be used by one skilled in the art toreplicate the Cup used to induce analgesia using any digital to analogconverter.

Design of Waveform

The low frequency pulsed magnetic fields are comprised of a plurality ofintermittent waveforms. The waveform is designed to look like thecorresponding electromagnetic waveform of the target tissue. Forexample, if the target tissue were a part, or parts, of the brain thenthe waveform would correspond to the energetic activity of those parts.If an electroencephalogram (EEG) could record that activity then thewaveform would mimic the EEG. As seen in FIGS. 1, 2, 3 and 4 thewaveform is not sinusoidal as this waveform was designed to affectcritical functions that do not rely on sinusoidal waveforms. Feature 1 ais a rise to a maximum and feature 1 b is designed to stimulate thefiring of axons in the tissue type of interest. Feature 1 c is a builtin delay to reduce the probability of neuronal excitation as thewaveform ends.

Latency Period

After each waveform or between successive waveforms there is a delay, alatency period. This delay is progressively set to increase, ordecrease, in length with time. This effectively modulates, in time, thefrequency of appearance of the waveform. The specific lengths andprogression of the Cnp waveforms are related to the target tissue. Withrespect to the central nervous system (CNS) for example, there are anumber of characteristic frequencies which relate to: a) frequenciesspecific to the area of the brain; b) frequencies associated withcommunication/connection between different brain regions; and c)frequencies and phase offsets associated with the co-ordination ofdifferent brain regions for a specific function. Now, although thewaveform has been designed to stimulate neuronal activity for a specificregion, electrical activity of a region of the CNS will vary betweenindividuals, and over time, within an individual. Therefore, to target afunction the frequency of presentation of the waveform should match thefrequency of the target. However, the target is varying within afrequency bandwidth. These CNS frequencies vary between approximately 7Hz to 300 Hz. (For example: 7 Hz corresponds to alpha rhythm; 10 Hzthalamic activity; 15 Hz autonomic time; 30 Hz intralaminar thalamus andtemporal regions associated with memory and consciousness; 40Hzconnection between hippocampal and amygdal temporal regions; 45 Hzhippocampal endogenous frequency; 80 Hz hippocampal-thalamiccommunication; 300 Hz motor control.) These frequencies have upperlimits due to neuronal electrical properties, that is: after a neuron“fires” it is left in a hyperpolarized state and cannot fire again untilit recovers. Therefore, Feature 2 (see FIG. 2) the latency period: a)allows neurons to recover so that when the waveform is reapplied theneuron can respond; and b) its length is set so that the frequency ofpresentation of the waveform matches or approximates the frequenciesassociated with the target.

Modulation of Latency Period

To change the electrical activity of the target tissue in the CNS, theCnp must “latch on” or more appropriately, entrain, to the appropriatefrequency and either slow it down or speed it up. The waveform itselfdoes not change substantially, rather, the frequency discussed hereincorresponds to the rate at which the waveform is presented and the rateat which electrical spikes occur in the target tissue. Generally, forthe CNS, as the frequency of neuronal activity is increased the amountof tissue involved per burst of activity decreases. Conversely, as thefrequency is decreased a greater amount of tissue is synchronized andrecruited throughout the CNS. For example, a) greater speed of cognitiveprocessing can be associated with increased rates; b) if the rate isdecreased significantly in humans or animals with epileptic-typedisorders so much tissue can be recruited that seizures will occur.Therefore, the ramping up or ramping down of the rate of presentation ofthe waveform will: a) ensure that at least at some time the applied andendogenous rates will be matched (provided of course that the initialrate is greater than the endogenous if the purpose is to reduce theendogenous rate or lower if the purpose is to increase the endogenousrate); and b) “pull down” or “push up” the endogenous rate.

Refractory Period

As a result of the application of the Cnp the synchrony of theelectrical activity of the target can be disrupted. Before theapplication of another Cnp can be effectual the tissue must recover itssynchrony. It is allowed to do so by providing a refractory periodbetween application of successive Cnps where the length of therefractory period is determined by the target. For example, if the Cnpsare applied to a target in humans which is associated with “awareness”,then the target will recover only after the awareness anticipation timeis exceeded (e.g. 1200 ms). Another example would be the application forthe same target, but in rodents without significant awareness, in whichcase the refractory period could be reduced to 400 ms. If the Cnps areto be applied for long periods of time per day, e.g. hours. then therefractory periods should be increased to 10 seconds to avoid possibleimmunosuppression. Immunosuppression has been show to occur when the CNSis stimulated chronically and this may be minimized if the refractoryperiods of this stimulation are increased to more than 7 seconds.

Variability in Features

It must be pointed out that the Cnp features are related to theunderlying physiology and that endogenous frequencies vary betweenindividuals and within an individual. Therefore, there is tolerance onthe feature specifications for any Cnp designed for a specific target.For example, in the analgesia pulse shown in FIGS. 1 and 2, the featurescan be varied somewhat and the outcome will remain similar due tobiological variations in the target. As well, as more and more islearned about biological interactions, the Cnp can be modified to takeadvantage of the new knowledge to make the Cnp even more specific.

Amplitude and Direction of Application

The amplitude of the Cnp to DC offset, and its direction of application(e.g. linearly polarized vs. circularly polarized vs. isotropicallypolarized), is dependent on the magnetic field detection mechanismwhich, may very well differ from one target to another. We haveexperimentally demonstrated that the amplitude of the Cnp can varysignificantly and that the Cnp is still effective provided the featuresremain constant for a specific application (Thomas et al, 1997).

Specifically, if magnetic fields are directly detected there will be awindow of amplitudes and the possible need of a DC offset to the Cnp forit to be effective. Further, the relative direction of the DC offset andthe time-varying portion of the Cnp is important. If the detectionmechanism is indirect, that is, induced currents, then an inducedcurrent feature, such as feature 1 d in FIG. 1 may be added to thewaveform of the Cnp. This preferably would be a feature with a highvalue of dB/dt with frequency components beyond those detectable by thetarget (i.e. for the CNS, greater than approximately 500 Hz) butdesigned to increase the induced EMF in the target. For magnetic fieldsdetected indirectly, a DC offset is ineffective but direction of theapplied Cnp may be important as a time changing magnetic field willinduce the greatest EMF in conductive tissue which projects a maximumarea normal to the direction of the Cnp. We have experimentally verifiedin a limited experimental trial that for some applications the effect isindependent of the DC offset.

The present invention is not at the magnetic field detection level, butrather in the coupling of a specific low frequency pulsed magnetic fieldto the target tissue. The Cnp design philosophy is not altered if thedetection mechanism is different for different targets. Rather, the Cnpis used in two “flavours”, one for direct detection and the other forindirect detection. Theoretically, it may be possible to produce the Cnpwaveform using other physical entities besides magnetic fields, such asflashing light, electrical fields, acoustic waves and peripheralstimulation of nerve receptors. However, extremely low frequency (ELF)magnetic fields remain the method of choice since they penetrate tissuewith minimal attenuation and since their amplitude can be spatiallydefined largely independent of the target. Hence, they are not limitedto specific targets. For example, sound is largely limited to auditorynerves, light to optic nerves and electric fields to conductive entrypoints such as the roof of the mouth. Also, the bandwidth of receptionmay be too low such as that defined by the “flicker fusion rate” of thevisual system. Nevertheless, Cnps may be used in the future with otherstimulation methods to increase target specificity.

Delivery-Exposure Systems

Exposure systems which produce variable magnetic field amplitude overthe subject's anatomy would be preferred in situations where theendogenous frequencies and waveform of the target overlap with othertissue which could produce unwanted “side-effects”. Magnetic resonanceimaging (MRI) gradient tube and gradient coil technology can be easilyadapted to produce such spatial variant Cnp exposures which can vary inboth magnetic field amplitude and direction. Therefore, it is better tohave two sets of volume coils for each of the three dimensions. One setwould produce the DC offset eg. Helmholtz configuration (Prato et al,1996) which would be needed if the detection mechanism is a resonancekind. The second would be used to define magnetic field gradients eg.Maxwell configuration (Carson and Prato, 1996)

This type of exposure system would be ideal for acute and chronicexposures in which the subject can stay in one position, e.g. treatmentof pain while the subject is in bed. For mobile subjects, such volumecoil configurations would not be possible and delivery would preferablybe through the use of surface coils either singly, as say on the surfaceof the body, or around the neck or as a Helmholtz pair placed on eitherside of the knee. In this configuration the magnetic field amplitudedecreases rapidly from the surface coil and matching of target andmagnetic field without exposing other tissue to an effective Cnp becomesmore challenging.

Applications of Cnps Analgesia

The applicants have reported that complicated pulsed magnetic fields(Cnps) have a pain inhibitory (analgesic) effect. In one embodiment ofthe present invention, the designed Cnps can both increase the analgesiceffect of an injection of an opiate, eg. Morphine, or actually induce alevel of analgesia similar to a moderate dose of morphine. This has atremendous benefit for the potential of drug-free pain treatment whichis highly desirable.

Opioid receptors are responsible, among many other functions, for themediation of pain. Increase in exogenous/endogenous opioids can induceanalgesia The applicants have shown that single sinusoidal ELF magneticfields can attenuate opioid induced analgesia. The applicants haverecently demonstrated that the detection mechanism responsible for thisresponse to ELF magnetic fields is a resonance model, that is, directdetection (Prato et al, 1996).

Since increases in opioid induced analgesia, rather than decreases,would have therapeutic value, and since induction of analgesia by ELFmagnetic fields would have even greater value the applicants havedeveloped a simple pure sinusoidal waveform specification that wouldinduce mild analgesia. As shown in FIG. 6 the increase was modest(20-30%). However, when the applicants designed a Cnp to induceanalgesia the effect was made much larger (FIG. 6).

The analgesia Cnp used and its magnitude Fast Fourier Transform areshown in FIG. 7. This in fact is the Cnp shown as FIGS. 1 and 2. This“analgesic pulse” can be used to: a) increase opioid induced analgesia;and b) significantly induce analgesia. In addition, it is now known thatanalgesia is only partially opioid mediated and that another analgesiccomponent is present. This additional component corresponds to themodulation of another target tissue or system, as yet unidentified. Thisis probably due to the more general nature of this Cnp, and that theentire animal was exposed to identical magnetic fields. The power in thefrequency was in three bands: 4-16 Hz; 22-26 Hz; and 28-52 Hz. The wholebody of the animals (land snail, Cepaea nemoralis) were exposed and thepurpose was to slow down activity in the brain structures which have ahigh concentration of opioid receptors and are responsible for theawareness of pain with frequencies in the range of 28-52Hz. Note, thatwhen a random pulse was used, in which frequency analysis indicatedconstant power in all frequencies between 0-166Hz, the induction ofanalgesia was not seen, indicating the specificity of even this generalCnp. The slowing up and disruption of function in such biological sitesin the snail equivalent to the CNS should have profound effects beyondthe induction of analgesia. In fact when whole rats with a pre-existingcondition of status epilepticus were exposed to this waveform, theresult was increased seizure activity. As previously discussed, whenfrequencies of waveform firings are reduced, more tissue is recruited.In this extreme case, sufficient CNS tissue was recruited in theelectrically labile rat exposed to this Cnp resulting in increasedseizure activity.

This Cnp pulse can be made more specific by treating subjects, likehumans, only over specific CNS structures or by incorporating moreselectively designed waveforms.

The applicant's have previously demonstrated that a short acute exposureto a specific weak extremely low frequency pulsed magnetic field (Cnp)can induce significant partly opioid-mediated analgesia in the landsnail, Cepaea nemoralis. In the first studies individual groups ofsnails, Cepaea nemoralis, were pre-injected with either the generalopioid antagonist naloxone or specific antagonists (μnaloxazine,β-funaltrexamine, δ naltrindole, ICI-174, 864 or ic nor-binaltorphimineopioid peptide specific antagonists), their respective injectionvehicles or received no injection and then were exposed for 15 minutesto a Cnp or a sham condition. The snails were then tested for responselatency on a hotplate (40° C.). There were no significant differences inpre-exposure response latencies, or in sham exposure response latencies,and hence, the individual groups were combined as seen in FIG. 8. Allgroups showed a significant degree of induced analgesia as inferred byan increase in response latency; however, the general p and 8 opioidantagonists significantly reduced, but did not block, the Cnp inducedanalgesia.

The time course of Cnp induced analgesia in these snails was alsoinitially investigated. Individual groups of snails were exposed to Cnpsfor either 15 or 30 minutes and then tested immediately, at 15, 30 and60 minutes after the Cnp exposure for response latency on a hotplate(40° C.). While there was no significant difference between the 15 and30 minute exposures, as compared to the sham exposure, there was asignificant degree of induced analgesia up to and including 60 minutepost exposure (FIG. 10).

The effect of Cnp induced analgesia has now been examined for thedevelopment of tolerance to daily repeated acute exposures of 15 or 30minute duration. Also examined was the effect of acute cross-toleranceto the d opioid receptor directed against the DPDPE enkephalin. Theresults of this study show that brief (15 or 30 min) exposure to aspecific pulsed magnetic field (Cnp) has antinociceptive or “analgesic”effects in the land snail Cepaea nemoralis. The magnitude and durationof this Cnp induced analgesia was reduced, though not blocked, followingrepeated daily exposures, in a manner indicative of the partialdevelopment of tolerance. Both associative (learning related) andnon-associative (pharmacological related) processes were suggested to belinked with the expression and reduction of the analgesic effect of thisspecific pulsed magnetic field (Cnp) following repeated exposures.Presentation of novel environmental cues could ameliorate the expressionof this tolerance and nearly re-instate the level of acute Cnp exposureinduced analgesia. These results are consistent with, and extend, priorfindings of specific pulsed magnetic fields, including the Cnp, havingbehavioral actions in invertebrate and vertebrate systems. These resultsalso substantiate and extend prior reports that the effects of ELFmagnetic fields on analgesia and likely other behavioral andphysiological responses can be modified with repeated brief dailyexposures.

Exposure for either 15 or 30 min to the Cnp resulted in a significantincrease in the latency of response of Cepaea to an aversive thermalsurface, indicative of the induction of analgesia. The magnitude of thisanalgesia was related to the length of exposure suggesting a possibleduration or dose-related effect of the Cnp. In previous studies it wasshown that the Cnp induced analgesia is not a generalized orstress-related effect of exposure to magnetic fields. Other similardesigns of pulsed magnetic fields were shown to have no significanteffects on either basal nociceptive sensitivity or opioid-inducedanalgesia [Thomas et al., 1997]. In addition, simple sinusoidalextremely low frequency magnetic fields (<300 Hz) have been shown eitherto attenuate or weakly augment opioid-mediated analgesia depending onthe specific magnetic field exposure characteristics. In the present, aswell as prior studies, repeated sham exposures (zeroed or normal Earthmagnetic field) and repeated determinations of response latencies, hadno significant effects on nociceptive sensitivity.

The significantly greater magnitude of analgesia induced by the 30exposure to the Cnp as compared to the 15 minute exposure is consistentwith the findings that the inhibitory effects of an acute magnetic fieldexposure on opiate analgesia are affected by both the duration ofexposure and the intensity of the magnetic field. Results of more indepth investigations, however, have also shown that the magnitude of theinhibitory effects does not scale linearly with either the frequency oramplitude of the ELF magnetic field [Prato et al., 1995].

Substantial evidence exists for the presence of multiple endogenousopioid inhibitory systems. Both naloxone-reversible ‘opioid’ andnatoxone-insensitive ‘non-opioid’ forms of analgesia have been indicated[Rothman 1996] and are apparently phylogenetically conserved andexpressed in both rodents and Cepaea [Kavaliers et al., 1983]. In priorinvestigations with Cepaea, it was established that the Cnp inducedanalgesia was of a mixed opioid and non-opioid nature [Thomas et al.,1997; Thomas et al., 1997(in press)]. The analgesic effects of the Cnpwere reduced, but not blocked, by the prototypic opiate antagonist,naloxone, and the d opioid receptor directed antagonists, ICI 174,842 ornaltrindole-5′-isothiocyanate (5′-NTII) (Table 2 and Thomas et al., 1997(in press)). However, the analgesic responses were unaffected bypretreatment with the kappa opioid directed receptor antagonist,norbinaltorphimine. This lack of a complete blockade of the Cnp inducedanalgesia by the opioid antagonists indicates that “non-opioid” as wellas opioid mediated mechanisms are associated with the effects of theCnp. The neurochemical mechanisms mediating this non-opioid analgesiaremain to be determined.

Typically chronic repeated administrations of opiates result in thedevelopment of tolerance, such that the analgesic effects initiallyproduced by substance such as morphine show a progressive decline inintensity until they are indistinguishable from the responses of controlanimals. Similar patterns and characteristics of morphine tolerance havebeen established to occur in Cepaea and rodents [Kavaliers et al., 1983;Kavaliers et al., 1985]. Here, it was determined that after 6-9 days ofdaily 15 or 30 min exposures to the Cnp, tolerance developed to theopioid mediated component of the induced analgesia. The pattern ofresponse and time course is similar to that for the development oftolerance to antinociceptive effects of opioid peptides and opiateagonists in Cepaea and rodents. The level of analgesia attained after6-9 days of daily exposure to the Cnp was similar to that recorded insnails treated with either naloxone or specific δ opioid receptordirected antagonists and followed by a single Cnp exposure. In addition,the snails that had received the daily exposures to the Cnp displayed asignificantly reduced sensitivity to the analgesic effects of thespecific 5 opioid agonist. DPDPE. This is suggestive of at least apartial generalization of tolerance (i.e. cross-tolerance) to the opioidcomponent of the Cnp. Determinations of the nociceptive responses ofsnails that have become tolerant to DPDPE and are subsequently exposedto the Cnp are necessary to explore more fully the extent of thisgeneralization and the expression of cross-tolerance between Cnp and δopioids.

In the present experiments there was little evidence of a reduction inthe level of the “non-opioid” mediated analgesia induced by repeatedexposures to the Cnp. The analgesia induced by the 15 min and 30 min Cnpexposures was reduced to a similar level. This raises the intriguingpossibility that increased duration of the Cnp may selectively augmentthe opioid mediated analgesia while leaving a relatively constant basalnon-opioid mediated component. It also suggests that various componentsof this specific Cnp may differentially affect the expression andneurochemical substrates of opioid and non-opioid analgesia.

There have been only limited considerations of the development oftolerance to naloxone-insensitive non-opioid analgesia. These studieshave revealed either relatively low or no development of tolerance tonon-opioid analgesia This is not, however, completely limited tonon-opioid analgesia, as weak tolerance has also been reported to theantinociceptive effects of certain opioid activating factors in rodents.There is also no apparent cross-tolerance between opioid and non-opioidanalgesia, with it having been speculated that the presence of opioidanalgesia may even preclude the development of tolerance to non-opioidanalgesia [Rothman 1996]. Similarly, it is possible that the presence ofnon opioid analgesia may affect the expression of opioid systems andlimit the expression of complete cross-tolerance as suggested here withDPDPE (FIG. 16).

It should also be noted that tolerance is considered to be bestdemonstrated by a shift in the dose-response indicative of the need fora higher dose to produce a consistent drug effect. In the present study,tolerance is inferred from the decrease in analgesia produced by dailyrepeated 15 or 30 min exposures to the Cnp. The lack of supportingevidence for a definitive linear dose-dependent effect of Cnp, alongwith the similar reductions in analgesic effects of the 15 and 30 minCnp exposures, precludes examination of shifts in dose responses.

Opiate tolerance has been proposed to involve both associative andnon-associative components. In prior investigations it was shown thatafter the termination of drug treatment, Cepaea that were rendered fullytolerant to morphine exhibited dependence and withdrawal symptoms,including hyperalgesia, that are considered to be consistent withnon-associative mechanisms [Tiffany et al., 1988].

Non-associative tolerance is considered to represent an effect arisingsolely from drug exposure. Tolerance is considered to result in partsimply from cellular adaptations produced by repeated drug stimulationof some physiological system such as a particular receptor or secondmessenger cascade.

Opioids have stimulatory as well as the more conventionally studiedinhibitory effects on neurotransmission that are accepted as themechanisms underlying analgesia. There is accumulating evidence thatthese stimulatory effects may also be associated with the development ofopioid tolerance. In this regard, daily acute exposures of Cepaea to ELF60 Hz magnetic fields were shown to result in hypoalgesic or analgesiceffects consistent with the antagonism of the excitatory hyperalgesiceffects of endogenous opioids.

There is also evidence that particular transmitter systems may functionto counteract opioid effects and mediate some aspects of tolerance. Inthis view, tolerance may not only result from decreased opiate efficacy,but also enhanced “anti-opiate” influences. The putative anti-opioidpeptide, orphanin FQ or nociceptin, which exerts its effects through anovel oiphan, opioid-like receptor, and has been recently implicated intolerance, has been shown to affect nociceptive responses in Cepaeathrough NMDA associated mechanisms. Intriguingly, orphanin FQ has alsobeen recently suggested to be involved in opioid mediatedelectro-acupuncture-induced analgesia [Tian et al., 1997].

Tolerance has also been shown to involve associative learning. Animals,including Cepaea, repeatedly receiving morphine in a consistent,distinctive environment are much more tolerant to the analgesic andthermic effects of morphine than when tested in a different, novel,environment. In the present study this “environmental specificity” wasdemonstrated for the opioid mediated analgesic effects of the Cnp.Snails that were exposed to the Cnp while in a novel environmentdisplayed an apparent reversal of tolerance, their analgesic responsesbeing similar to that of individuals receiving single acute Cnpexposures (FIG. 15).

A variety of factors, including ELF magnetic fields, have been shown tofunction as salient environmental specific cues and affect thesubsequent expression of tolerance. This raises the possibility that theCnp itself may at least partially serve as a cue for tolerancedevelopment. This may contribute in part to the apparent lack of acomplete “cross-tolerance” to the analgesic effect of the Cnp to the δopioid agonist DPDPE.

Associative, environmental or situation specific tolerance has beenexplained through classical conditioning, [Tiffany et al., 1981]although habituation involving both associative and non-associativecomponents has also been proposed [Baker et al., 1985]. According to theconditioning model the distinctive context has become a conditionedstimulus that elicits associative tolerance.

In the present study it was found that similar patterns of tolerancedeveloped whether the snails received nociceptive testing every day oronly on the first and last days. This suggests that associative factorsrelated to determining the thermal response latencies (i.e. hotplatetesting) of the snails did not play a major role in the development oftolerance. This also minimizes the likelihood that tolerance arises fromcues associated with the nociceptive assessment. This is consistent withthe results of a number of investigations of opiate tolerance inrodents, as well as morphine tolerance in Cepaea.

Recent studies have focused on the possible neurochemical mechanismsinvolved in associative tolerance. Investigations with laboratory ratshave suggested that neurotensin and possibly other neuropeptidesimplicated in memory may have a role in the mediation of associativetolerance [Girsel et al., 1996]. This does not preclude a role for otherneuronal and second messenger systems that have been implicated inlearning in both molluscs and rodents, and been shown to be sensitive tovarious types of magnetic fields.

A number of possible mechanisms have been proposed for the biologicaleffects of magnetic fields [Kavaliers et al., 1994; Prato et al., 1995].Among these, resonance models have predicated both increases anddecreases in opioid analgesia along with effects at specificfrequencies. These actions have been suggested to have effects oncalcium and potassium ions and various messenger systems [Kavaliers etal., 1996; Kits et al., 1996; Prato et al., 1996; and Kavaliers et al,1996], that are associated with the mediation of opioid actions andlearning related processes. All of these could contribute to the Cnpinduced expression of analgesia and decline in the opioid component withrepeated exposures.

Vestibular System

The use of Cnp pulses appears to be valuable for affecting variousvestibular components of mammals. With respect to humans, Cnps can bevery valuable for the alteration of standing balance. Disruptions of thebalance system such as motion sickness may possibly be treated with theuse of Cnps without adverse side-effects such as nausea or sleepiness.

The Cnp shown in FIG. 3 was used to target the vestibular system inrodents (activity study; amplitude 100 mT), in deer mice (conditionedtaste aversion study; amplitude 100 mT), and in rats (conditioned tasteaversion study; amplitude 1-4 mT). The Cnp shown in FIG. 5 was alsopiloted in humans (balance study; amplitude 10-60 mT). Note that FIG. 3and FIG. 5 differ only in the length of the refractory period. In humansthe refractory period (Feature 4 in FIG. 1) was 3 times longer than forrodents. The reason is that awareness lasts 3 times as long for humanswho extrapolate each awareness period (approximately 400 ms) withcognitive function to three such periods (approximately 1200 ms).

The rate or waveform presentation was modulated from higher frequenciesto lower frequencies and two different waveforms were used. FIG. 5 showsthe magnitude Fourier transform of the Cnp, i.e. it is a magnitudespectrum of the positive frequencies and the maximum frequency possiblewas set to 500 Hz by the digital representation of the Cnp at aseparation of 1 ms. Note, that FIG. 5 indicates that the power in thespectrum is at three major frequency ranges: 100-125; 125-240; 325-410.The high frequencies were needed since the vestibular system is a motorfunction and, therefore, has endogenous CNS frequencies of the order of300Hz.

Two different waveforms were used to represent the electromagneticactivity of the vestibular system. This was necessary to provide aminimum resolution time (1 ms) at the highest frequencies. Initially, atwo lobe waveform was used and then when the waveform rate wassufficiently reduced and the latency sufficiently long a five lobewaveform was used as it was believed to better mimic the underlyingelectrical activity of the target tissue.

Modulation of Anxiety

Severe anxiety has been shown to accompany depression. A Cnp has nowbeen designed which significantly alters anxiety related responses inmice.

A Cnp designed to produce vestibular disturbance in deer mice produced amarked increase in activity (activity index =total of escape behaviorssuch as climbing attempts, jumps, centerline crosses) during a 10 minuteCnp exposure as compared to a 10 minute sham exposure (FIG. 17). The 100μT Cnp exposure and sham condition were given while the animals werecontained in a Plexiglass open-field box. The Cnp exposure also produceda significant decrease in the duration of grooming behaviors (FIG. 25).

Modulation of Behavioral Activities

Deer mice were exposed to a specific Cnp designed to interact with thevestibular system to characterize the effects of Cnp on behavioralactivities. Individual deer mice were exposed to the Cnp or controlconditions (sham or 60 Hz) for a 10 minute period while being videotapedand various behaviors were monitored. It was concluded from this studythat specific pulsed magnetic fields (Cnps) may be designed to affectselectively a variety of behaviors. Acute exposures (5 min) aresufficient to produce a significant behavioral effect (FIGS. 19-25).Cnps were seen to affect rearing behaviors and general activity such asclimbing and centerline crosses compared to the control groups. Theseresult demonstrate that Cnps can be used to alter a variety ofbehavioral activities.

Taste Aversion

Field studies have indicated that deer mice, Peromysciis Maniculatus,developed long lasting avoidance of poisoned baits, whereas results ofan early laboratory investigation of conditioned taste aversion (CTA)suggested the formation of taste aversions that extinguished rapidly.The applicants have examined in one set of experiments the acquisitionand extinction of a conditioned taste aversion (sucrose paired withLiCl) in male and female deer mice. In another set of experiments, theapplicants have examined the acquisition and extinction of conditionedtaste aversion using sucrose alone in Wistar rats and in deer mice. Theapplicants also examined the effects of specific Cnps on taste aversionlearning. Together, the results of these studies (FIGS. 26-29)demonstrate that Cnp can be used to modify taste aversion in deer miceand in Wistar rats.

A Cnp designed to interfere with vestibular processing was tested foraversive effects in two independent trials of conditioned tasteaversion, or taste aversion learning. In one experiment, Wistar rats(N=24) that were exposed to the specific Cnp for one hour at beingprovided with a novel food item (sucrose solution) consumedsignificantly more sucrose solution when tested three days afterexposure, as compared to sham exposed animals (F_(1.23)=5.99, P=0.023,Eta²=0.22). In another experiment, deer mice (Peromyscus maniculatus)(N=43) were exposed to either the specific Cnp or a sham condition forone hour. After exposure, the deer mice were given access to water andapple juice simultaneously and the ratio of apple juice to total volumeconsumed (apple juice+water) was recorded. The deer mice exposed to theCnp consumed significantly more apple juice than did the sham exposedmice (F_(1.43)=3.95, P=0.05). Though the exposure systems used in thetwo experiments were vastly different, the same specific Cnp was used.In both cases neither induced an aversion to the novel food. Results ofprior investigations had shown that the specific Cnps were capable ofinducing other specific behavioral affects in those species. Experimentone utilized a single coil (72 turns of 30 WG) wrapped around analuminum (1.3m×1.1 m) cage rack (100-700 μT Cnp exposure, normal Earthearth magnetic field sham (Michon et al, 1996)). The exposure system forexperiment two consisted of three pairs of nested orthogonal Helmholtzcoils (Prato et al, 1996) (100±0.1 μT, 3-D±0.1 μT zeroed Earth fieldmagnetic sham).

The results of the studies using sucrose and LiCl showed thatreproductive male and female deer mice developed a rapid conditionedtaste aversion to a sucrose solution that was paired with lithium. Therewas a complete extinction of the aversion after 4-5 days with noevidence of a residual aversion 10 days later which is a contrast to thelonger lasting aversions generally evident in laboratory rats. Therewere also sex differences in the conditioned taste aversion with maledeer mice displaying a longer lasting aversion and slower extinctionthan females.

The Cnp exposure did not elicit a conditioned taste aversion, but ratherit reduced the neophobic responses of males to a novel taste and sexdifference in baseline taste preferences. Further experiments conductedat Laurentian University also revealed that the specific Cnp similarlyreduced neophobic responses and aversions to novel food items inlaboratory rats.

Overall, these findings indicate that the effect of the specific Cnp, inat least a taste aversion paradigm, is dependent on the“characteristics” of the magnetic field, not the exposure system,amplitude, geographical location or species tested.

Learning

All behaviors, including learning, originate as a pattern of electricalactivity in the brain. Using specific Cnps, specific behaviors can bealtered inferring that specific areas of the brain can be selectivelyaffected. Previous studies using Cnps have shown alterations inbehaviors such as language, memory, suggestibility, mood andunderstanding. It is anticipated that combinations of specific Cnps willresult in predictable alterations of memory and learning.

Epilepsy

The use of Cnps has great potential to treat epilepsy safely, a seriousproblem associated with brain trauma.

Depression

The potential to treat depression with Cnps is enormous, in bothclinical and model terms (Baker-Price and Persinger, 1996). Also,related disorders such as ‘seasonal affective disorder’ may prove to besusceptible to Cnp treatment. It has been envisioned that the equipmentrequired for this Cnp treatment would be portable, about the size of a‘Walkman’, and have earphone sized head coils.

The designed pulsed magnetic fields (Cnps) of the present invention canbe used effectively to treat a variety of physiological andpsychological conditions in a safe and effective manner. Any livingorganism including humans and animals can be subjected to the Cnps ofthe present invention. By safe and effective as used herein is meantproviding sufficient potency in order to decrease, prevent, ameliorateor treat a a physiological or neurological disorder affecting a subjectwhile avoiding serious side effects. A safe and effective amount willvary depending on the age of the subject, the physical condition of thesubject being treated, the severity of the disorder, the duration oftreatment and the nature of any concurrent therapy.

The subjection of a subject to effective Cnps exposures of the presentinvention is defined as an amount effective, at dosages and for periodsof time necessary to achieve the desired result. This may also varyaccording to factors such as the disease state, age, sex, and weight ofthe subject, and the ability of the Cnps to elicit a desired response inthe subject. Dosage or treatment regima may be adjusted to provide theoptimum therapeutic response. For example, several divided doses ortreatments may be administered daily or the dose may be proportionallyreduced as indicated by the exigencies of the therapeutic situation.

The Cnps of the present invention may be subjected to a mammal alone orin combination with pharmaceutical agents or other treatment regimes.

EXAMPLES Example 1 Materials and Methods Animals

Snails were collected from old field sites in London, Ontario which didnot have any overhead or underground electric transmission lines (<0.01μT ambient magnet fluctuation). Snails were then individually numberedby applying a small identifying mark on the apex of the shell usingnon-toxic colored fingernail polish. The individually numbered animalswere held in a terrarium (ambient fluctuating magnetic fields <0.4 μT)under indirect natural and fluorescent, lighting at an approximate 12 hlight/ 12hr dark cycle (LD=12:12 L=250 μW/cm²), at 20±2° C., withlettuce available ad lib.

Assessment of Nociception

As the activity of gastropods is affected by their state of hydration[Smith 1987], all snails were allowed to fully hydrate under a saturatedatmosphere at 20±2° C. before being tested. Individual fully-hydratedsnails were placed on a warmed surface (“hotplate”40±0.2° C.) and thelatency of their “avoidance” of the thermal stimulus, was determined.The avoidance behavior was a characteristic elevation of the anteriorportion of the fully extended foot, the behavioral endpoint being thetime the foot reached maximum elevation [Dyakonova et al., 1995]. Afterdisplaying this aversive, or more appropriately, “nociceptive” response[ Kavaliers et al., 1983], individual snails were removed from thethermal surface. An increase in response latency may be interpreted asan antinociceptive or “analgesic” response [Thomas et al., 1997]. Thehotplate, which does not produce any magnetic fields, consisted of analuminum waterjacket with a stainless steel top (33×33cm) with waterpumped through it from a circulating water bath.

Experimental Apparatus

Groups of 15 snails were placed in translucent polypropylene containers(12 cm square, 5 cm high) in the center of three mutually orthogonalHelmholtz coils (1.2 m for the coil that generated a vertical field and1.1 m and 1.0 m for the coils that generated horizontal fields). Detailsof the coils and amplifiers are provided in Prato et al.[Prato et al.,1996]. A computer driven 8-bit resolution digital to analog converter(S. Koren, Neuroscience Research Group, Laurentian University, Sudbury,Ontario) was used to produce the pulsed waveforms. Magnetic fields weremeasured with a fluxgate magnetometer (model FGM-3DI) and a fieldmonitor (model ELF-66D), both Walker Scientific, Worcester, Mass.

Magnetic Field Exposure Conditions

The 15 and 30 min magnetic field exposures consisted of a specific lowfrequency pulsed magnetic field (Cnp) (FIG. 9) set to 100 μT peakamplitude in the vertical direction.

Sham exposures consisted of a three-dimensionally (3-D) zeroed Earthmagnetic field (Helmholtz coils tuned to oppose the Earth's magneticfield to within ±0.1 μT horizontal component=14.7 μT, verticalcomponent=43.3 μT.) Results of prior investigations [Thomas et al.,1997; Thomas et al., 1997 (in press)] had established that there were nosignificant differences in the response latencies of 3-D zeroed Earthmagnetic field sham exposed snails and those that were exposed to anambient Earth magnetic field sham condition.

Example 2 Materials and Methods for Taste Aversion Studies Animals

Male and female sexually mature deer mice (20-25 g and approximately 5months of age) were housed in mixed-sex sibling groups (3-5 animals pergroup) in polyethylene cages provided with cotton nesting material andBeta chip bedding. Food (Purina Rat Chow) and water were available adlibituni. The reproductive mice (males scrotal, females cyclic) wereheld under a reproductively stimulatory (Desjardins et al. 1986; Nelsonet al. 1992), long day, 16 h light: 8 h dark cycle (light 0600-2200 hr)at 20+/−2° C. The laboratory bred deer mice (15-20 generations) werefrom a population of wild caught animals originally present in theinterior of British Columbia (Canada) near Kamloops (50° 45′N, 120°30′W).

Additional characteristics of this wild and laboratory population areprovided in Innes and Kavaliers (1987).

Experimental Procedures

There were five phases in this first experiment: a habituation phase; aconditioning phase: a postconditioning recovery phase, an extinctionphase and a re-test phase.

Habituation Phase

Males (n=21) and females (n=22) were individually housed for 10 dayswith food and tap water, in standard drinking bottles, available adlibitum. For 4 days the mice were water deprived overnight (darkperiod). Each morning they were given two drinking tubes containing tapwater and the total amount of water consumed over 90 min. was recordedFor the remainder of the day tap water in standard drinking bottles wasavailable ad libitum. The drinking tubes consisted of 15 ml graduatedpolypropylene conical tubes (Falcon 2092, Becton Dickinson, LincolnPark, N.J., USA), with screwable caps that were fitted with stainlesssteel water spouts with ball bearing. With these drinking tubes fluidintake could be accurately determined to 0.25 ml. Individual bodyweights were recorded at night immediately after the removal of thewater bottles and in the day directly after the drinking tubes wereremoved.

Conditioning Phase

On Day 5 overnight water deprived mice were given the two drinking tubeseach of which contained a 0.3 M sucrose solution. After 90 min. thetotal sucrose intake was recorded and mice were immediately injectedintraperitoneally (i.p.) with 20 ml/Kg of either a 0.15 M LiCl solutionor 0.9% isotonic saline solution. After injection the water bottles werereturned and the mice were then left undisturbed until the evening whentheir bottles were removed and the mice were weighed. Male and femalemice were randomly assigned to the UCI and saline groups.

Post Conditioning Recovery Phase

For two days after conditioning (days 6 and 7) mice were kept on theirnightly water deprivation schedule. In the mornings they were presentedthe two drinking tubes with tap water for 90 min. after which water fromthe standard drinking bottles was provided ad libitunt. Body weightswere recorded twice daily as previously described.

Extinction Phase

On days 8-11 individual mice were presented-two drinking tubes, onecontaining tap water and the other one holding the 0.3 M sucrosesolution in the morning drinking period. Water and sucrose solutionintakes were recorded for 90 min. after which tap water from standardwater bottles was available ad liitum. The position of the two drinkingtubes was varied: half of the mice in each experimental group (LiCl andNACI) had water on the right and the other half had sucrose on theright. To correct for possible individual preferences, the position ofthe water and sucrose tubes were reversed on subsequent days. Deer micewere weighed twice daily.

Recovering Phase

After extinction of the conditioned taste aversion the male and femaledeer mice were left undisturbed for 10 days with ad libituwn access tofood and tap water.

Retesting Phase

Individual male and female mice were replaced on the overnight waterdeprivation schedule. For two days the water deprived mice were given inthe morning two drinking tubes, one containing water and the other 0.3 Msucrose and their total fluid intakes were determined over 90 min. Afterthis 90 min. period they were provided with ad libitum access to thestandard water bottles.

Total fluid intakes were analyzed by a two way individual analysis(MANOVA) with sex (two levels; mails and females) and treatment (twolevels; LiCl and NaCI) as between-subject factors and intake as arepeated-measure within-subjects factor (eleven levels; Habituation(four days), conditioning (pairing and 2 post injection days),extinction (four days)). In order to evaluate the effects of sex andtreatment on the fluid intakes on each day mean comparison were planneda priori in the MANOVA model. Since fluid intake displayed a Poissoniandistribution the data were square-root transformed before analysis. Asthere were some zero intakes, 0.50 was added to all values beforetransformation.

Preference data from extinction and re-test phases were expressed as thepercent of sucrose (arcsin transformed) consumed in the total fluid.These preference data were analyzed by a two way MANOVA, with sex (twolevels; males and females) and treatment (two levels; LiCl and NaCI) asbetween subject factors and percent of sucrose as a repeated-measurewithin subjects factor (six levels: 4 extinction days+2 re-test days).In order to evaluate the effects of sex and treatment on sucrosepreference for each experimental day, mean comparisons were planned apriori in the MANOVA model.

Body weights were analyzed by a two way MANOVA with sex (2 levels; malesand females) and treatment (2 levels; LiCl and NaCI) as between-subjectfactors and intake as a repeated-measure within-subjects factor (21levels). In order to evaluate the effects of sex and treatment on themice body weights for each expeomental day, mean comparisons wereplanned a priori in the MANOVA model. All hypothesis tests used a=0.05as the criterion for significance.

Magnetic Field Generation

In the magnetic and sham field exposure conditions mice were placed in aPlexiglas box in the centre of three mutually orthogonal Helmoltz coils(1.2 m diameter for the coil that generated a vertical and 1.1 m for thecoils that generated horizontal fields; details of the coils andamplifiers are provided in Prato et al. (1994). A computer driver with a8 bit resolution digital to analog converter produced the pulsedwaveforms. Magnetic fields were measured with a fluxgate magnetometer(model FGM - 3DI) and a field monitor (model ELF - 66D; both WalkerScientific, Worchester, Mass. USA).

Magnetic Field Exposure Conditions

The magnetic field exposures consisted of a specific low frequencypulsed magnetic field set to 100+/−0.1 μT; peak amplitude in thevertical direction.

Sham exposures consisted of a three dimensionally (3-D) zeroed earthfield (Helmoltz coils tuned to oppose the earth's field to within+1/−0.1 μT; horizontal component=14.7 μT, vertical component=43.3 μT).

Example 3 Experimental Procedures, Opioid Experiments Experiment 1

Each day for 9 consecutive days, at midphotophase, separate groups (n=15per group, N=120) of hydrated snails were exposed to either the specificpulsed magnetic field or sham magnetic field for either 15 or 30 min. Onday 10, the exposure conditions were reversed for each group, with shamanimals receiving the Cnp and Cnp exposed animals receiving shamexposure. Response latencies of the snails were determined prior to(Pre), immediately after (0) and 15, 30 and 60 min after exposure. Oneindividual carried out the Cnp and sham exposures while a secondexperimenter, in a separate room, determined the response latencies.Results of previous investigations had established that tolerance to theanalgesic effects of morphine in Cepaea was evident after 7 days ofdaily repeated acute treatments [Kavaliers et al., 1983].

Experiment 2

Each day for 9 consecutive days, at midphotophase, other separate groups(n=15 per group, N=60) of hydrated snails were exposed to either thespecific Cnp or sham magnetic field for 15 or 30 min and then (exceptfor days I and 9) immediately returned to their home container. On daysI and 9 response latencies of the snails were individually determinedprior to (Pre), immediately after (0) and 15, 30 and 60 min afterexposure, after which they were returned to the home container.

Experiment 3

After 9 days of daily repeated acute Cnp or sham exposure (15 min) andassessment of nociception (pre, 0, 15, 30, 60 min), other groups ofsnails (N=30) were exposed (day I0) to their respective exposureconditions while held in a novel environment. The novel environmentconsisted of a modified version of the previous polypropylene exposurecontainer. Pieces of coarse garnet sandpaper were fitted and glued tothe top and bottom inside of the container and then rinsed with carrotjuice (whole blended carrot). Carrot is assumed to be a novel food item,as the laboratory housed snails were not exposed to this food item atany time. In addition, other naive snails (N=60) were exposed to eithera Cnp or sham condition while housed in either the C, “normal” or“novel” exposure environment. The novel environmental condition had noeffect on the magnetic field exposure characteristics.

Experiment 4

After 9 days of daily repeated acute Cnp or sham exposure (30 min) andassessments of nociception (pre, 0, 15, 30, 60 min), individual snails(N=60) were injected (day 10 with either DPDPE (0,05 μg/1.0 μl saline,Research Biochemicals, Natick, Mass.) or 0.9% saline vehicle (1.0 μl).Nociceptive sensitivity was determined prior to and 15, 30, 60 min afterinjection. This dose of DPDPE was established in a prior study to elicitan analgesic response comparable in magnitude to that observed after asingle acute Cnp exposure [Thomas et al., 1997 (in press)]. Allsolutions were injected with a 2.0 μl microsyringe (No. 75, Hamilton,Nev.) in either the vicinity of, or directly in, the mantle cavity intothe haemocoel. Injections were made on the basis of 1.0 g body mass. Thebody mass of snails, without shells, range from 0.7 to 1.3 g.

Experiment 5

The Thermal response latencies of snails receiving Cnp and treatmentswith opiate antagonists.

Snails were injected with either the prototypic opiate antagonist,naloxone (1.0 μg/1.0 μl saline), the specific δ antagonist,naltrindole-5′-isothiocyanate (5′-NTII, 0.l μg/1.0 μl saline) or salinevehicle (1.0 μl) prior to being exposed for 15 min to the specific Cnp.Other groups of snails received either acute sham magnetic field, acuteCnp exposure (15 min) or daily repeated (9days) acute Cnp exposure.There were no significant differences in response latencies betweenopiate antagonist treated animals (naloxne, 5′-NTII) and those receivingCnp exposures (acute daily exposure for 9 consecutive days). Acute Cnpexposure produced significantly greater response latency than all othergroups (Tukey's HSD, P<0.05).

Experiment 6

The effect of Cnp exposure on behavioral activities in deer mice.

Individual deer mice (Peromyscus maniculatus) (N=46) were exposed for 10min (analyzed in 5 min segments), while being videotaped, to either; anormal Earth magnetic field, 14.7 μT horizontal and 43.3 μT verticalsham condition, a 3-D zeroed Earth magnetic field (+/−0.1 μT) shamcondition, 60 Hz (100 +/−0.1 μT vertical) sinusoidal magnetic field or aspecific Cnp (100 +/−0.1 μT peak) condition. The exposure chamberconsisted of a 33 cm Plexiglass cube held within three pairs of nestedorthogonal Helmholtz coils (1.2m×1.1 m×1.0 m) (Prato et al., 1996). Thevideotapes were then analyzed by an experimenter blind to the exposureconditions. Various behavioral activities were recorded (center-linecrossings, climbing attempts, rearing and duration of grooming episodes)indicating that the pulsed Cnp exposed deer mice had a significantlyincreased level of activity compared to the normal Earth magnetic field,3-D Earth magnetic field sham and 60 Hz MF exposure conditions. Therewere no significant differences in activity between the normal Earthmagnetic field, 3-D zeroed Earth magnetic field sham or 60 Hx MFexposure conditions.

Statistical Analysis

Data were analyzed with multivariate, repeated measures, one-way andtwo-way analyses of variance (ANOVA) using The Statistical Package forSocial Sciences (SPSS 7.0). Post-hoc analyses were carried out usingTukey's HSD test. All hypotheses tests used α=0.05 as the criterion forsignificance.

Example 4 Experimental Results, Oploid Studies Experiment 1

Acute single exposure to the Cnp elicited a significant(F_(4.115)=268.59, P<0.001, Eta²=0.90) increase in response latencyindicative of the induction of analgesia at 0, 15. 30 and 60 min postexposure. The 30 min exposures induced a significantly greater amplitudeof analgesia than did the 15 min exposure at 0, 15 and 30 min postexposure F_(4.113)=4.71, P<0.01, Eta²=0.14) (FIG. 10). In both cases,maximum analgesia was elicited at 0-15 min post-exposure withsignificantly lower response latencies at 30 and 60 min post exposure.Repeated daily exposures to the Cnp resulted in a significant reductionin the levels of analgesia. By the third day, no significant differencesin the increases in response latency were elicited by the 15 and 30 minexposures (FIGS. 11A, 11B and 12A, 12B).

The analgesic effects of daily repeated acute exposure to a Cnp magneticfield were highly significant (F_(1.55)=2856.4, P<0.001, Eta²=0.95)(FIG. 12A), consistently producing a significant increase in responselatency. Repeated analysis of variance revealed a significant reductionin daily induced analgesia (F_(8.48)=86.29, P<0.001, Eta² 0.94) (days 1to 9); and a significant reduction in the duration of analgesic effect(F_(4.52)=230.66, P<0.001, Eta²=0.95) (pre-exposure and 0, 15, 30, 60min after daily Cnp exposure) (FIG. 12A). Although the amplitude of Cnpinduced analgesia was significantly reduced after the repeated dailyexposure, a significant analgesia was present after each Cnp exposure(Tukey's HSD, P<0.05) (FIGS. 12A, 12B). Maximum reductions in analgesiawere evident after day 6 of exposure to the Cnp, with no significantfurther reduction of response latency on subsequent days. There were nosignificant changes in either pre-exposure basal response latencies orthe nociceptive responses of the sham exposed snails (FIGS. 12A, 12B,12C). Reversal of the exposure conditions on day 10 produced asignificant shift in response latency (F⁴⁵²=110.8, P<0.001, Eta²=0.90).The Cnp exposure induced significant analgesia in the previously shamexposed snails while the snails now exposed to the sham condition showedno significant increase in response latency (FIG. 13A, 13B).

Results of previous studies [Thomas et al., 1997 (in press) and Table 2] had established that pre-treatment with either the prototypic opiateantagonist, naloxone, or the specific δ receptor directed antagonist,5′-NTII, significantly reduced, but did not block, the analgesic effectof the Cnp. The saline vehicle had no significant effect on responselatency. These inhibitory effects of the opiate antagonists on theamplitude and time course of Cnp induced analgesia were comparable tothe reduction in response latency and levels of analgesia that wereobtained by repeated daily exposure (6-9 days) to the Cnp (Table 2).

Experiment 2

Snails that were exposed to the Cnp daily for either 15 or 30 min, buttested for nociceptive responses only on days I and 9, showed asignificant reduction in Cnp induced analgesia (F_(1.110)=3144.4,P<0.001, Eta²=0.93). The extent of this reduction was not significantlydifferent from that seen in snails that received daily acute Cnpexposures and nociceptive assessments. There were no significantdifferences in pre-exposure or sham exposure latencies.

Experiment 3

The presence of a novel environment on day 10 of the repeated dailyexposures to the Cnp caused a significant increase in the level of Cnpinduced analgesia (F_(1.27)=250.6, P<0.001, Eta²=0.90)(FIG. 15). Theamplitude of the analgesic response evident in the novel environment wassignificantly greater than on day 9 of the daily repeated exposures tothe Cnp (F_(1.27)=6.98, P<0.01, Eta²=0.24). The elevated responselatencies evident following exposure to the Cnp in the novel environmenton day 10 were not significantly different from those of day 1 of therepeated exposures under normal environmental conditions. Other groupsof naive snails receiving an acute exposure to the Cnp or sham condition(15 min), while in either the normal or novel environment, showedelevated response latencies indicative of Cnp induced analgesia(F_(1.58)=248.76, P<0.001, Eta²=0.90). There were no significantdifferences in the levels of analgesia induced in the two environmentalconditions (F₁₅₈=1.31, P>0.50, Eta²=0.04). There were no significantdifferences in pre-exposure or sham exposure latencies.

Experiment 4

Treatment with the specific δ opiate agonist, DPDPE, produced asignificant analgesic effect in snails that had received daily repeatedsham magnetic field exposures for 9 days (F_(1.59)=86.97, P<0.001,Eta²=0.87) (FIG. 16). This analgesic effect was similar to thatpreviously observed in naive unexposed snails treated with DPDPE (0.05μg 1.0 μl) [Thomas et al., 1997 (in press)]. Snails that received acute(15 min) exposures to the Cnp and were injected with DPDPE alsodisplayed a significant analgesic response, with increased responselatencies at 15 and 30 min post-injection. However, the magnitude ofthis analgesia was significantly lower than that displayed by the shamexposed DPDPE treated snails (FIG. 16). The level of analgesia inducedby DPDPE in the snails that had received 9 days of daily repeatedexposures to the Cnp was similar to the analgesic effect elicited by theCnp exposure on day 9 of the repeated exposures. The saline vehicleinjection (1.0 μl) had no significant effect on response latencies.There were no significant differences in pre-exposure or sham exposurelatencies.

Example 5 Experimental Results Taste Aversion Studies Total FluidIntakes

The total fluid intakes across the various experimental phases are shownin FIG. 27. Overall there was a significant interaction of treatment bysex by time (F_((10.390))=2,09; P=0.02). By the 4th day of thehabituation phase all of the overnight water deprived males and femalesconsumed similar amounts of tap water during the 90 min. presentation ofthe drinking tubes. Likewise, an the pairing day males and femalesassigned to both treatment groups drank the same amount of the novelsucrose solution. The amount of sucrose consumed was not different fromthat of the tap water on the last day of the habituation phase.Similarly, the two sexes and treatment groups of deer mice did notdiffer in the total water intakes in the post conditioning (recovery)days as well as on the first two days of the extinction phase. On thirdday of the extinction phase females overall drank significantly marethan males (F_(1,39))=8.42; P=0.006). This sex difference was highlysignificant for the NaCl treated mice (F_((1.18))=5.50; p=0.02). thoughnot for the LiCl treated mice (F_((1.19))=3.32; p=0.09). On the fourthday of the extinction phase there were no significant male-female orgroup differences in total fluid intakes.

On the re-test days the MANOVA showed that total fluid intakes offemales were significantly greater than those of males on both days(main factor sex RE-TEST-1:F_(1.39))=6.17; P=0.017/main factor sexRE-TEST-2: F_((1.39))=5.93; P=0.019).

Percent Sucrose Intake

The percent of sucrose consumed by the deer mice during the extinctionand the retest phases is shown in figure The overall analysis showed asignificant main effect of treatment (F_((1.108))=0.45: P=0.003). asignificant interaction of treatment×time (F_((3,108))=3.481; P=0.02)with the interaction treatment×time×sex approaching significance(F_((3.108))=2.609; P=0.05). The MANOVA showed that on the first andsecond days of the extinction phase LiCl treated male and female miceingested a significantly lower percent of sucrose (first day: 13%;second day. −28%) than vehicle treated males and females (first day:58%; second day: 66%, with no significant sex difference) mice (Firstextinction day: F_((1.36))=1 9,00; P=0.001/Second extinction day:F_((1.36))=17,35; P=0.0002). On the third extinction dav only LiCltreated males still showed a significant reduction in the percent ofsucrose solution ingested (males: F_((1.18))=4,12- P=0.049/females:F_((1.18))=1.96; ns). On this day the percent of sucrose intake of theLICL treated males was 46%. while that of vehicle treated males was 70%of sucrose. Conversely, the sucrose preference of females was equallyhigh in both the LiCl (61%) and vehicle (76%) groups. By dav 4 also themales had recovered from the conditioned sucrose aversion and thepercent of sucrose drunk was not significantly different between sexesand treatments (overall mean).

On the re-test phase, ten days after recovering from the taste aversionall of groups displayed a similar marked preference (80%) for thesucrose solution, indicating that the greater total fluid intakedisplayed by females did not reflect a sex difference in tastepreferences (FIG. 28).

Body Weights

There were no significant sex differences or effects of treatment onbody weights.

Total Intakes

The total fluid intakes across the various experimental phases are shownin FIG. 27 Overall there was a significant main effect of sex(F_(1.195))=4.28; P=0.045) as well a significant interaction of sex xintake in time (F_((5.195))=2.43; P=0.036). The MANOVA showed that onthe pairing day, when apple juice and the magnetic/sham field werepresented, males and females did not differ in their total intakes ofapple juice.

On the two days after the magnetic/sham field exposure (sucrose andwater presented), there was a significant interaction sex×fieldcondition (POST-1: F_((1.39))=4.16; P=0.048/POST-2: F_((1.39))=4.53;P=0.043). Mean comparisons revealed hat only sham exposed femalesdisplayed a greater intake than sham-exposed males (FIG. 28). Thiseffect was stronger on the first day post-magnetic/sham field exposurewhen sham-exposed females drank more than either magnetic field-exposedfemales and or males of both exposure groups (Sham Females v s Shammales: F_((1.19))=9.55; P=0.004/Sham Females vs Pulse females:F_((1.20))=7.61; P=0.009/Sham females vs Pulse males. F_((1.19))=9.19;P=0.004). On the second day following the magnetic/sham exposure femalesof the sham group still consumed a significantly greater amount of fluidthan the sham-exposed males (F_((1.19))=4.21; P=0.047). On the applejuice re-presentation day males and females, in both magnetic and shamexposed groups, did not differ in their total fluid intake.

Percent of Sucrose

All of groups displayed a similar marked preference (80%) for thesucrose solution, indicating that the greater total fluid intakedisplayed by females did not reflect a sex difference in tastepreferences (FIG. 26).

Percent of Apple Juice

The percent of apple juice consumed by male and female deer mice on thethird day after exposure to the magnetic/sham field are shown in FIG.28. There was a significant main effect of treatment (F_((1.39))=5.28:P=0.02). Mean comparisons revealed that the effect of the pulse on thedeer mice reaction to the novel fluid item was different in male andfemale. Magnetic field-exposed female deer mice consumed the samepercent of apple juice as sham exposed females (F_((1.20 ))=0.32; ns).Conversely sham exposed males consumed a significantly lower percent ofapple juice than either the magnetic field exposed males(F_((1.19))=7,765-, P×0.008) or the magnetic field-exposed females(F_((1.19))=4.87; P=0.03). However, they did not consume less thansham-exposed females (F_((1.19))=1.98: ns). FIG. 28 shows that magneticfield exposed mice of both sexes consume a high percent of apple juice(males: 79%, females: 75%). In the sham exposed group only femalesshowed a preference for the apple juice (68%). while males consumedequal quantities of apple juice and tap water (51% of apple juice),suggesting that the magnetic field exposure increased the initial lowpreference of males for the novel taste.

After being re-tested all mice, that at this stage all displayed amarked preference for the sucrose solution, were then used to examinethe effects of a specific pulsed magnetic field an taste preferences.There were three phases in this second experiment. conditioning phase(pairing of the magnetic field with a novel taste; apple juice);post-conditioning phase (sucrose preference) and post-magnetic/shampairing apple juice preference-determination.

Body weights were not determined as the results of previous experimentshad established that the experimental procedures had no significanteffect an body weight.

Conditioning Phase: Novel Fluid (Apple Juice) and Magnetic/Sham FieldExposure.

On the morning of the first day water deprived mice were given twodrinking tubes both containing pure unsweetened apple juice (McIntosh,Master's Choice, Canada). Apple juice was a novel fluid which in pilotstudies deer mice had been shown to readily consume. Apple juice intakeswere measured over 90 min. The mice were then immediately placed in thenovel holding cage (four mice per time, two males and two females) thatwas quickly (30 s) moved into the magnetic field apparatus where theywere exposed for 60 min. to either the pulsed magnetic or sham field.Each exposure cage was divided in four separate compartments by opaquePlexiglas partitions that prevented the individual mice from seeing eachother. Thus four mice per time (2 males and 2 females) were exposed tothe same field condition. Mice were assigned to the magnetic/shamexposed groups in a quasi-randomized manner. From each of the LiCl/NaClgroups of experiment 1 half of the males and half of the females wereexposed to the magnetic field, while the remaining animals underwent thesham exposure. The order of the exposures were quasi-randomized, with asham field exposed group following each magnetic field exposure group of4 mice. The box was washed with hot water and unscented soap betweenexposures. After being exposed to the magnetic/sham field the mice werereturned to their home cages with ad libitum food and water. Water wasremoved overnight.

Post Conditioning Phase Percent of Sucrose Intake

On the two days following the magneticlsham field exposure mice wereplaced an the overnight water deprivation schedules. In the morningsthey were presented with two drinking tubes, one containing 0.3 Msucrose and the other tap water. Their intakes were determined for 90min. after which mice were provided with ad libitum access to tap water.

Post Pairing Percent of Apple Juice Intake

On third day after magnetic/sham field exposure overnight water depriveddeer mice were presented with two drinking tubes, one containing waterand the other apple juice and their fluid intakes over 90 min. weredetermined. After this 90 min. period of time the mice were providedwith ad libitum access to tap water. The position of the tubes wasquasi-randomized: half of the mice in each experimental group(Magnetic/sham field exposed; males/females) had water on the right andthe other half had apple juice on the right.

Total fluid intakes across all the experimental days were analyzed by atwo way MANOVA, with sex (two levels; males and females) and treatment(two levels; Magnetic and Sham field) as between-subject factors andintake as a repeated-measure within subjects factor (four levels). Inorder to evaluate the effects of sex and treatment on the fluid intakeon each experimental day, mean comparisons were planned a prior-i in theMANOVA model. Since fluid intake displayed a Poissonian distribution thedata were square-mot transformed before analysis. As there were somezero intakes, 0.50.was added to all values before transformation.

The sucrose preference data were expressed as the percent of sucroseingested (arcsin transformed) and were analyzed by a two way MANOVA,with sex (two levels; males and females) and treatment (two levels;Magnetic and Sham field) as between subject factors and percent ofsucrose ingested as a repeated-measure within subjects factor (twolevels, two days after magnetic/sham field exposure). In order toevaluate the effects of sex and treatment on the sucrose preference oneach experimental day, mean comparisons were planned a priori in theMANOVA model.

The apple juice preference data were expressed as percent of apple juiceconsumed (arcsin transformed) and were analyzed by a two way ANOVA withsex (two levels; males and females) and treatment (two levels; Magneticand Sham Field) as between subjects and percent of apple juice as thedependent variable. In order to evaluate the effects of sex andtreatment on the fluid preference in each experimental day, meancomparisons were planned a priori in the ANOVA model.

Although preferred embodiments have been described herein in detail, itis understood by those skilled in the art that variations andmodifications may be made to the present invention without departingfrom the spirit and scope thereof as defined by the appended claims.

TABLE 1 0 −53 −50 0 96 127 0 −92 −53 0 −128 0 0 0 −128 0 0 0 12 −128 0 0108 72 0 −56 −53 127 −116 0 0 0 −116 0 0 0 24 −116 127 0 120 72 0 0 −12872 −104 0 0 0 −104 0 0 0 36 −92 72 12 127 −53 0 0 −116 72 −50 0 0 0 −500 0 0 48 −56 72 24 127 −53 0 0 −92 −53 0 12 0 0 0 0 0 0 60 0 −53 36 −128−128 0 0 −56 −53 127 24 0 0 127 12 0 0 72 0 −53 48 −116 −116 0 0 0 −12872 36 0 0 72 24 0 0 84 12 −128 60 −104 −92 0 0 0 −116 72 48 0 0 72 36 00 96 24 −116 72 −50 −56 0 0 0 −92 −53 60 0 0 −53 48 0 0 108 36 −92 84 00 0 0 0 −56 −53 72 0 0 −53 60 0 0 120 48 −56 96 127 0 0 0 0 0 −128 84 00 −128 72 0 0 127 60 0 108 72 0 0 0 0 0 −116 96 0 0 −116 84 0 0 127 72 0120 72 0 0 0 0 0 −92 108 0 0 −92 96 0 0 −128 84 12 127 −53 0 0 0 0 0 −56120 0 0 −56 108 0 0 −116 96 24 127 −53 0 0 0 0 0 0 127 0 0 0 120 0 0−104 108 36 −128 −128 0 0 0 0 0 0 127 0 0 0 127 0 0 −50 120 48 −116 −1160 0 0 0 0 0 −128 0 0 0 127 0 0 0 127 60 −104 −92 0 0 0 0 0 0 −116 0 0 0−128 0 0 127 127 72 −50 −56 0 0 0 0 0 0 −104 0 0 0 −116 0 0 72 −128 84 00 0 0 0 0 0 0 −50 0 0 0 −104 0 0 72 −116 96 127 0 0 0 0 0 0 0 0 0 0 0−50 0 0 −53 −104 108 72 0 0 0 0 0 0 0 127 12 0 0 0 0 0 −53 −50 120 72 00 0 0 0 0 0 72 24 0 0 127 12 0 −128 0 127 −53 0 12 0 0 0 0 0 72 36 0 072 24 0 −116 127 127 −53 0 24 0 0 0 0 0 −53 48 0 0 72 36 0 −92 72 −128−128 0 36 0 0 0 0 0 −53 60 0 0 −53 48 0 −56 72 −116 −116 0 48 12 0 0 0 0−128 72 0 0 −53 60 0 0 −53 −104 −92 0 60 24 0 0 0 0 −116 84 0 0 −128 720 0 −53 −50 −56 0 72 36 0 0 0 0 −92 96 0 0 −116 84 0 12 −128 0 0 0 84 480 0 0 0 −56 108 0 0 −92 96 0 24 −116 127 0 0 96 60 12 0 0 0 0 120 0 0−56 108 0 36 −92 72 0 0 108 72 24 0 0 0 0 127 0 0 0 120 0 48 −56 72 0 0120 84 36 0 0 0 0 127 0 0 0 127 0 60 0 −53 0 12 127 96 48 0 0 0 0 −128 00 0 127 0 72 0 −53 0 24 127 108 60 12 0 0 0 −116 0 0 0 −128 0 84 12 −1280 36 −128 120 72 24 0 0 0 −104 0 0 0 −116 0 96 24 −116 0 48 −116 127 8436 0 0 0 −50 0 0 0 −104 0 108 36 −92 0 60 −104 127 96 48 0 0 0 0 0 0 0−50 0 120 48 −56 0 72 −50 −128 108 60 12 0 0 127 12 0 0 0 0 127 60 0 084 0 −116 120 72 24 0 0 72 24 0 0 127 0 127 72 0 0 96 127 −104 127 84 360 0 72 36 0 0 72 0 −128 84 0 0 108 72 −50 127 96 48 0 0 −53 48 0 0 72 0−116 96 0 0 120 72 0 −128 108 60 0 0 −53 60 0 0 −53 0 −104 108 0 12 127−53 127 −116 120 72 0 0 −128 72 0 0 −53 0 −50 120 0 24 127 −53 72 −104127 84 0 0 −116 84 0 0 −128 0 0 127 0 36 −128 −128 72 −50 127 96 0 0 −9296 0 0 −116 0 127 127 0 48 −116 −116 −53 0 −128 108 0 0 −56 108 0 0 −920 72 −128 0 60 −104 −92 −53 127 −116 120 0 0 0 120 0 0 −56 0 72 −116 072 −50 −56 −128 72 −104 127 0 0 0 127 0 0 0 0 −53 −104 0 84 0 0 −116 72−50 127 0 0 0 127 0 0 0 0

TABLE 2 Experimental Response Latency Condition sec ± sem (n) Sham 4.9 ±0.2 (45) Acute Cnp 10.5 ± 0.4 (15)  Repeated Cnp 7.2 ± 0.3 (15) Cnp +vehicle 9.4 ± 0.3 (48) Cnp + Naloxone 7.7 ± 0.3 (21) Cnp + 5′-NTII 7.3 ±0.3 (22) sem (Standard Error of the Mean)

REFERENCES

1. Kavaliers, M.; Ossenkopp, K.-P. (1991) Opiold systems and magneticfield effects in the land snail, Cepaea nemoralis. Biol. Bull.180:301-309.

2. Prato, F. S., Ossenkopp, K-P., Kaveliers, M., Sestini, E. A. &Teskey, G. C. (1987) Attenuation of morphine-induced analgesia in miceby exposure to magnetic resonance imaging: Separate effects of thestatic, radiofrequency and time-varying magnetic fields. Mag. Res.Irnag. 5, 9-14.

3 Betancur, C., Dell'Omo, G. and Alleva E., (I 994) Magnetic fieldeffects on stress-induced analgesia in mice: modulation by light,Neurosci. Lett., 182 147-150.

4. Kavaliers, M.; Ossenkopp, K -P.; Prato, F. S.; Carson, J. (1994)Opioid systems and the bilogical effects of magnetic fields. In Frey AH(ed): On the nature of electromagnetic field interactions withbiological systems. Austin, RG Landis Co. pp181-190.

5. Del Seppia, C.; Ghione, S.; Luchi, P.; Papi, F. (I 995) Exposure tooscillating magnetic fields influences sensitivity to electricalstimuli. 1: Experiments on pigeons. Bioelectromagnetics 16:290-294.

6. Papi, F.; Ghione, S.; Rosa, C.; Del Seppia, C.; Luschi, P. (1995)Exposure to oscillating magnetic fields influences sensitivity toelectrical stimuli. 11: Experiments on humans. Bioelectromegnetics.16:295-300.

7. Papi, F.; Luschi, P. & Limonta, P. (1991) Orientation-disturbingmagnetic treatment affects the pigeon opioid system. J. exp. Biol. 160,169-179.

8. Kavaliers, M., Eckel, L. A. & Ossenkopp, K -P (1993) Brief exposureto 60 Hz magnetic fields improves sexually dimorphic spatial learningperformance in themeadow vole, Microtus pennsvivanicus. J comp. Physiol.A 173, 241-248.

9. Kavaliers, M., Ossenkopp, K -P., Prato, F. S. et at. (1996) Spatiallearning in deer mice: sex differences and the effects of endogenousopiods and 60 Hz magnetic fields. J comp. Physiol A (In the press).

10. Polk, C. (1992) Dosimetry of extremely low frequency magneticfields. Bioelectrornagnetics Supp. 1, 209-235.

11. Weaver, J. S. & Astumian, R. D. (1990). The response of living cellsto very weak electric fields; the thermal noise limit. Science, Wash.247, 459-462.

12. Kirschvink, J. L. & Walker, M. M. (1985). Particle sizeconsiderations for magnetite-based magnetoreceptors. In Magnetitebiomineralisation and magnetoreception in organisms: a new biomagnetism(ed. J. L. Kirschvink, D. S. Johnes & B. J. MacFadden), pp. 243-256. NewYork:Plenum Press.

13. Prato, F. S., Kavaliers, M. & Carson, J. J. L.(1996a) Behaviouralevidence that magnetic field effects in the land snail, Cepaeanemoralis, might not depend on magnetite or induced electric currents.Bioelectromagnetics 17, 123-130.

14. Rothman, R. B.(1996) A review of the role of anti-opioid peptides inmorphine tolerance and dependence. Synapse. 12:129-136.

15. Kavaliers, M.; Hirst, M. (1983) Tolerance to the morphine-influencedthermal response in the terrestrial snail, Cepea nemoralis.Neuropharnacology. 22(11):1321-1326.

16. Thomas, A. W.; Kavaliers, M.; Prato, F. S.; Ossenkopp, K -P. (1997)Antinociceptive effects of a pulsed magnetic field in the land snail,Cepaea nemoralis. Neurosci Lett. 222:107-110.

17. Thomas, A. W.; Kavaliers, M.; Prato, F. S.; Ossenkopp, K -P. (inpress, 1997) Pulsed magnetic field induced “analgesia” in the landsnail, Cepaea nemoralis, and the effects of μ, 67 , and κ opioidreceptor agonistslantagonists. Peptides.

18. Thomas, A. W.; Persinger, M. A. (1997) Daily post-training exposureto pulsed magnetic fields that evoke morphine-like analgesia affectsconsequent motivation but not proficiency in maze learning in rats-Electro-and Magnetobiology. 16(1):33-41.

19. Kavaliers, M.; Hirst, M.; Teskey, G. C. (1983) A functional role foran opioid system in snail thermal behavior. Science. 220:99-101.

20. Kavaliers, M.; Ossenkopp, K.-P. (1985) Tolerance to morphine-inducedanalgesia in mice: magnetic fields function as environmental specificcues and reduce tolerance development. Life Sci. 37:1125-1135.

21. Tiffany, S. T.; Maude-Griffin, P. M. (1988) Tolerance to morphine inthe rat: associative and non-associative effects. Behav. Neurosci.102:434-443.

22. Tian, J -H.; Xu, W.; Zhang, W.; Fang, Y.; Grisel, J. E.; Mogil, J.S.; Grandy, D. K.; Han, J -S. (1997) Involvement of endogenous OrphaninFQ in electroacupuncture-induced analgesia. Neuroreport. 8:497-500.

23. Tiffany, S. T.; Baker, T. B.(1981) Morphine tolerance in rats:congreunce with a pavlovian paradigm. J Comp. Physiol. Psych.95:747-762.

24. Baker, T. B.; Tiffany, S. T. (1985) Morphine tolerance ashabituation. Psychological Reviews. 92-78-108.

25. Girsel, J. E. G.; Watkins, L. R.; Maier, S. F. (1996) Associativeand non-associative mechanisms of morphine analgesia tolerance areneurochemically distinct in the rat spinal cord. Psychopharmacology.128:245-255.

26. Prato, F. S.; Carson, J. L. L.; Ossenkopp, K.-P; Kavaliers, M.(1995) Possible mechanisms by which extremely low frequency magneticfields affect opioid function. FASEB. J. 9:807-814.

27. Kits, S. K.; Mansvelder, H. D.(1996) Voltage gated calcium channelsin molluscs: classification, CA²⁻ dependent inactivation, modulation andfunctional roles. Invertebrate Neuroscience. 2:9-34.

28. Prato, F. S.; Kavaliers, M.; Carson, J. L. L. (1996) Behavioralevidence that magnetic field effects in the land snail, Cepaeanemoralis. might not depend on magnetite or induced electric currents.Bioelectromagnetics. 1 7:123-130.

29. Smith, K. H., Jr. (1987) Quantified aspects of pallial fluid and itsaffect on the duration of locomotor activity in the terrestrialgastropod Triolopsis albolabaris. Physiol. Zool. 54:407-414.

30. Dyakonova, V.; Elofsson, R.; Carlberg, M.; Sakharov, D.(1995)Complex avoidance behavior and its neurochemical regulation in the landsnail, Cepaea nemoralis. Gen. Pharmacol. 26:773-777.

31. Michon A, Koren S A, Persinger M A (1996): Perceptual and MotorSkills 82:619-626.

32. Baker-Price L. A. and M. A. Persinger, (1996) Weak, but complexpulsed magnetic fields may reduce depression following traumatic braininjury. Perceptual and Motor Skills, 83, 491498.

33. M. Kaveliers, K.-P. Ossenkopp, F. S. Prato, D. G. L. Innes, L. A. M.Galea, D. M. Kinsella, T. S. Perrot-Sinal, (1996) Spatial learning indeer mice: sex differences and the effects of endogenous opioids and 60Hz Magnetic fields. J. Com. Physio A 179.

What is claimed is:
 1. A method for treating a disorder selected fromthe group of physiological, neurological and behavioral disorders, saidmethod comprising applying to a subject a specific low frequency pulsedmagnetic field (Cnp) having a plurality of intermittent waveforms, for atime effective to produce a desired effect in a target tissue, whereinsaid Cnp initially entrains the electrical activity of the target issueand as a result affects the endogenous electrical activity of saidtarget tissue.
 2. The method of claim 1, wherein said plurality ofwaveforms are configured with length and frequency relative to thetarget tissue.
 3. The method of claim 2, wherein said waveforms areconfigured to mimic generally the underlying electrical activity of thetarget tissue.
 4. The method of claim 2, wherein said plurality ofwaveforms have a built-in variable latency period.
 5. The method ofclaim 1, wherein said low frequency pulsed magnetic field (Cnp) isdesigned with a built in delay to reduce excitation in said targettissue.
 6. The method of claim 4, wherein said latency period isprogressively lengthened to reduce the burst firing rate of endogenouselectrical activity of said target tissue.
 7. The method of claim 4,wherein said latency period is moderated differently in sequentialwaveforms to simultaneously target a number of different tissues.
 8. Themethod of claim 1 wherein said low frequency pulsed magnetic field has afixed refractory period relative to said target tissue.
 9. The method ofclaim 1, wherein said method is used to treat disorders selected fromthe group consisting of pain, anxiety, balance, learning, tasteaversion, epilepsy and depression.
 10. The method of claim 1, whereinsaid Cnp used in the method is selected from the Cnps of FIG. 1, FIG. 3or FIG.
 5. 11. The method of claim 1 wherein the frequency and length ofsaid waveforms vary over time.
 12. A method for treating a disorderselected from the group of physiological, neurological and behavioraldisorders, said method comprising applying to a subject a specific lowfrequency pulsed magnetic field (Cnp) having a plurality of intermittentwaveforms, for a time effective to produce a desired effect in a targettissue and wherein the frequency of said waveforms decrease over time.13. The method of claim 1 wherein the frequency of said waveformsincrease or decrease over time.
 14. The method of claim 1 wherein saidwaveforms have fast rise times and are configured to stimulate firing ofaxons in said target tissue.
 15. The method of claim 1 wherein saidwaveforms define variable latency periods, said latency periods beingselected to reduce the probability of neural excitement as the waveformsend.
 16. The method of claim 1, wherein said waveforms have amplitudesand DC offsets selected in relation to the target tissue.
 17. The methodof claim 1, wherein said low frequency pulsed magnetic field (Cnp) isdesigned with a built in delay to reduce excitation in said targettissue.
 18. The method of claim 1, wherein a static magnetic fieldoffset is applied to said target tissue.
 19. The method of claim 1,wherein said method additionally comprises simultaneously applying aspecific low frequency non-magnetic pulsed field to said target tissue,said specific low frequency non-magnetic pulsed field being selectedfrom the group consisting of light, electrical fields, acoustic wavesand peripheral stimulation of nerve receptors.
 20. A method of treatingphysiological, neurological and behavioral disorders comprising the stepof subjecting target tissue to intermittent specific time varying lowfrequency magnetic fields for a duration effective to produce a desiredeffect, said intermittent magnetic fields being separated by refractoryperiods, and wherein said intermittent magnetic fields initially entrainthe electrical activity of said target issue and as a result affect theendogenous electrical activity of said target tissue.
 21. The method ofclaim 20, wherein said waveforms are configured to mimic generally theunderlying electrical activity of the target tissue.
 22. The method ofclaim 20 wherein the frequency and length of said waveforms vary overtime.
 23. The method of claim 20 wherein the frequency of said waveformsincrease or decrease over time.
 24. The method of claim 20, wherein saidrefractory periods are fixed at a duration relative to the targettissue.
 25. The method of claim 20, wherein said waveforms have fastrise times and are configured to stimulate firing of axons in the targettissue.
 26. The method of claim 20 wherein said wavefonns definevariable latency periods, said latency periods being selected to reducethe probability of neural excitement as the waveforms end.
 27. Themethod of claim 20, wherein said waveforms have amplitudes and DCoffsets selected in relation to the target tissue.
 28. A method oftreating physiological, neurological and behavioral disorders comprisingthe step of subjecting target tissue to intermittent specific timevarying low frequency magnetic fields for a duration effective toproduce a desired effect, said intermittent magnetic fields beingseparated by refractory periods and having waveforms configured relativeto the target tissue and wherein the frequency of said waveformsdecrease over time.